Many biological reactions which are potentially interesting for large scale in vitro industrial use are prohibitively expensive due to the requirement for costly coenzymes. The present invention describes an application of the enzyme reactor described in copending Ser. No. 026,380 which will allow complex coenzyme-requiring enzymic reactions to be economically scaled up as industrial processes. The invention lies in the inclusion of coenzyme-requiring enzyme, coenzyme, and a coenzyme regeneration enzyme system within the hydrogel layer of the reactor such that the coenzyme will not diffuse out or be depleted.
A constant supply of coenzyme for the enzyme reaction is assured by initially charging the hydrogel layer with an adequate supply of coenzyme. In operation of the enzymatic process, the coenzyme is prevented from diffusing out of the hydrogel either by a thin film polymer membrane which is impermeable to the coenzyme or by switching to a solvent in which the coenzyme is not soluble to carry substrate materials. The problem of coenzyme depletion is solved by including a second enzyme in the hydrogel layer which regenerates the used coenzyme, thus recycling it. The substrates for both the primary (coenzyme-requiring) and regeneration reactions are supplied in the circulating solvent. Reaction products from the reaction of substrate and the coenzyme-requiring enzyme are carried away by the solvent, from which they may be recovered. The coenzyme produced from the reaction of substrate and the regeneration enzyme remains within the hydrogel layer while any other reaction products from that reaction are carried away by the solvent.
Most common, industrial, enzymatic reactions are carried out in bulk reaction systems, ordinarily in batch reactions. The enzyme component which catalyzes the desired reaction is usually discarded as waste at the conclusion of the reaction. This occurs even though the enzyme functions as a catalyst and theoretically it is therefore possible to recover and recycle the enzyme at the conclusion of the reaction. Apart from the expense of recovery, it has often been found that the activity of recovered and recycled enzymes is degraded by the recovery procedures and in many cases undesirable or intolerable contaminants are introduced.
For many enzyme catalyzed processes, the desirability and need for continuous, as opposed to batch, processing and other features has led to extensive investigations of techniques and means for the immobilization of enzymes on supports of one kind or another. The most commonly employed procedure at present is glutaraldehyde immobilization by the formation of covalent bonds to the enzyme, which form the basis for cross-linking the product to a physical support. The support is most often a granular solid, although there have been numerous investigations and use of other forms of supports, including membrane supports, particularly to physically entrap enzymes within the pores of membrane structures, most often with the additional use of chemical immobilization. It has been observed, however, that in many systems enzyme activity is impaired or even completely lost as a consequence of interference of the covalent cross-linking with the reactive site of the enzyme. There have been observations which reveal that the temperature and pH optima are also altered by such procedures. In some circumstances, advantage may be taken of the changes in properties, but on the whole, it is desirable to provide a technique which retains the original properties of the enzyme to the greatest possible degree.
In other contexts, there have been investigations of systems for physical entrapment or encapsulation of enzymes. The objectives of these procedures have generally been to avoid the unfavorable consequences of covalent bonding immobilization procedures, while retaining the advantages thereof. These systems and approaches have met with limited success and acceptance for a variety of reasons. Among these are characteristics which result in the loss of direct and intimate contact between the enzyme and the substrate, because of the limited diffusion capacities of such materials and structures, with the attendant losses in production rate and efficiency, and the rather substantial cost penalties involved.
One approach to physical entrapment of enzymes has been to confine the material on or in a membrane structure, where the enzyme remains lodged while the substrate is flowed through the membrane. The resulting stream is processed to recover the product, and the substrate is recycled. By these techniques, the art has attempted to provide direct and intimate contact between the enzyme and the substrate, and by using commercially available membranes, the costs of this type of immobilization are kept to a reasonable level. These techniques have not met with acceptance, however, since the efficiencies of the system may be impaired in other ways. Notably, there is a trade-off between the permeability of the membrane, i.e., the resistance to flow of the substrate process stream, and the effectiveness of the containment of the enzyme. When the controlling or limiting pore size of the membrane is optimal for confining the enzyme, the hydraulic resistance to flow of the substrate containing stream is often unacceptably high. When the pore size is enlarged to a level more consistent with the flow rates required for reasonable through-put, there is an increasing risk of enzyme loss into the product stream. In some circumstances, the result is an inconvenient burden on the product purification, but in other circumstances, such results are intolerable.
Most enzyme reactions in biological systems are very efficient in terms of the energy and substrates consumed, primarily because of their specificity. Efficiency is essential for competitiveness with other organisms. Enzymes are complex macromolecules which are "expensive" for the organism to manufacture. The energy investment in the enzyme is justified because in its lifetime it can catalyze millions of reactions, each at a low energy expense, and generally produce products which would be impossible to create in a living environment without catalysis.
The activity of some enzymes are dependent only on their structure as proteins, while others also require one or more nonprotein components, called cofactors. Cofactors may be metal ions or organic molecules called coenzymes.
A coenzyme is a molecule which is essential for, but consumed in the catalyzed reaction. Like the enzyme, coenzymes are valuable molecules which are preferably conserved. Coenzymes are organic molecules and generally contain as part of their structure one or another of the vitamins, which are trace organic substances which are vital to the function of all cells. Nearly all of the water-soluble vitamins, including all of the "B" vitamins, biotin and lipoic acid, function as components of coenzymes.
Coenzymes for the most part serve as intermediate carriers of functional groups, specific atoms or electrons which are transferred in the overall enzymatic reaction. For example, some coenzymes transiently bind functional groups in the enzymatic transfer from one molecule to another, e.g., thiamin pyrophosphate which transfers aldehyde groups and the pyridoxine coenzymes which transfer amino groups. Other coenzymes frequently provide reducing or oxidizing power for the enzyme to use in the reaction, e.g., pyridine nucleotides, flavin nucleotides. Examples of some of the principal coenzymes and the types of enzymatic reactions with which they involved are shown in the following table, TABLE I:
TABLE I ______________________________________ Coenzyme Entity transferred ______________________________________ Nicotinamide adenine Hydrogen atoms (electrons) dinucleotide Nicotinamide adenine Hydrogen atoms (electrons) dinucleotide phosphate Flavin mononucleotide Hydrogen atoms (electrons) Flavin adenine dinucleotide Hydrogen atoms (electrons) Coenzyme Q Hydrogen atoms (electrons) Thiamin pyrophosphate Aldehydes Coenzyme A Acyl groups Lipoamide Acyl groups Coenzyme B.sub.12 1, 2 shift of hydrogen atoms Biocytin Carbon dioxide Pyridoxal phosphate Amino groups Tetrahydrofolate coenzymes One-carbon group transfer ______________________________________
When the coenzyme is very tightly bound to the enzyme molecule, it is usually called a prosthetic group, e.g., the flavin nucleotides (containing riboflavin or B.sub.2), coenzyme A (containing pantothenic acid), or the biocytin group of acetyl CoA carboxylase. In other cases, the coenzyme is loosely bound to the enzyme and essentially functions as one of the specific substrates for the particular enzyme, e.g., the pyridine nucleotides (containing niacin) and tetrahydrofolate (from folic acid).
In biological systems, every enzyme reaction which consumes coenzyme is linked to a reaction which generates coenzyme. If the coenzyme is tightly or covalently bound, these linked reactions, namely the enzyme reaction and the coenzyme regeneration reaction, take place on the same enzyme. If the coenzyme is loosely bound and functions as a substrate, then the linked reactions will take place on different enzymes. The linked reactions can either both produce needed products or a waste product may be generated.
Many enzymatic reactions which are dependent on cofactors, especially coenzymes, are of potential interest for large scale in vitro commercial and industrial use. Such use, however, is prohibitively expensive and costly, especially when loosely bound coenzymes are an essential component of the catalytic system. The necessary concentrations of these coenzymes along with the reaction substrates cannot be economically provided for in a large commercial scale. Accordingly, the present invention is directed to a method which would allow economical industrial use of enzymes which are coenzyme dependent, and involves the isolation of the coenzyme and the enzyme within the hydrogel layer, and the inclusion of a coenzyme regeneration system.